The appicant's overall goal is to determine the extent to which physiological changes in free calcium (Ca2+) and mitochondrial NADH contribute to the control of cardiac respiration. Since three mitochondrial dehydrogenases are activated by Ca2+ and increased sarcoplasmic Ca2+ stimulates mechanical work, it has been proposed that Ca2+ functions to increase both work and respiration in parallel. An increase in cytosolic Ca2+ is transmitted to mitochondria to increase matrix Ca2+, stimulating three enzyme activities; isocitrate, a- ketoglutarate and pyruvate dehydrogenases. In addition, the applicant obtained data to suggest that an increased mitochondrial Ca2+ stimulates the malate-aspartate shuttle for transport of NADH into mitochondria. The Ca2+- induced increased respiratory capacity and maintenance of ATP levels are thought to result from an increased and/or sustained level of mitochondrial NADH when oxygen consumption and NADH utilization are increased. With ratio surface fluorescence techniques, however, the applicant obtained data in the intact heart to suggest that mitochondrial NADH levels may not be a dominant factor in respiratory control. More recently, other studies have demonstrated that cardiac work causes a stimulation of the mitochondrial F1-ATPase by a process that may also be Ca2+-mediated. In this study, the applicant will use optical techniques to measure Ca2+ and mitochondrial NADH in the isolated perfused heart. By systematic control of these two parameters, he will determine the extent to which NADH participates in respiratory control and the effect of Ca2+ on this process. The applicant will also determine specifically the importance of cytosolic Ca2+ in this control in experiments designed to manipulate the Ca2+ gradient across mitochondria.